Potent Bioactivity of Endophytic Fungi Isolated from Moringa oleifera Leaves

Plant species are known to harbor large number of endophytes, which stays in plant tissues as symbionts. These endophytes secrete large array of bioactive compounds that have potency against certain diseases with no side effects. We have collected leaf samples of the Moringa oleifera plant from the Pakistan Forest Institute, Khyber Pakhtunkhwa, Pakistan for the isolation of beneficial endophytes. The strains isolated from the leaves of M. oleifera were coded with MOL and tested for antimicrobial, antifungal, germicidal, phytotoxic, insecticidal, cytotoxic, and anti-inflammatory activities. The isolates, MOL1, MOL16, MOL19, and MOL21, possessed antibacterial activity against Staphylococcus aureus, whereas MOL7 inhibited 55% of the growth of Escherichia coli. MOL3 inhibited the growth of E. coli, S. aureus, and Pseudomonas aeruginosa. The strains, MOL1 and MOL7, showed antifungal activity against Candida albicans and Saccharomyces cerevisiae, while the strains, MOL11 and MOL17, showed activity against Verticillium chlamydosporium. The isolates, MOL3, MOL7, MOL9, MOL15, MOL17, MOL18, and MOL19, inhibited the growth of Lemna minor (duckweed) at 100 μg/ml. MOL2 exhibited strong activity in the brine shrimp assay, while MOL1, MOL2, MOL5, MOL6, MOL12, MOL17, MOL19, and MOL20 showed insecticidal, and MOL3 demonstrated larvicidal and antileishmanial activity. The isolated potent endophytes were identified as Aspergillus, Penicillium, Fusarium, Tricoderma, Rhizoctonia, Mucor, Alternaria, Pestalotiopsis, Acremonium, and Cladosporium through morphological and microscopic characteristics of the colonies.


Introduction
Fungal endophytes live inside tissues of their host plants and had been reported for beneficial effects on their host. Endophytes became known to human knowledge since last century. However, there is also a hypothesis that the evolution of endophytes dates back to evolution of land plants [1]. Recently, endophytes are gaining interest as sources of biologically active secondary metabolites [2]. Exploration of the endophytic microflora is a step towards saving medicinally important plants from extinction. It encourages to har-vest medicinally important metabolites from endophytic microbiota instead of the host plant species [3].
Moringa oleifera commonly known as Drumstick, Ben tree, Magic tree, or Suhanjna (Urdu) is a tree having huge medicinal importance. Pharmacological studies showed that the ethanolic extract of M. oleifera leaves have antihypertensive (hypotensive) activity [4]. Similarly, alcoholic extracts of seed kernels of M. oleifera exhibited antiasthmatic activity [5], whereas the aqueous extract of leaves demonstrated antidiabetic activity [6]. In another study, the hepatoprotective activity was noticed in leaves and seeds [7] and anticancerous activity was noted in ethanolic extract of M. oleifera seeds [8,9].
Fungal endophytes are known to produce a large number of bioactive compounds that are beneficial for humans as well as plants [10][11][12][13][14][15]. Literature showed that the minimum inhibitory concentration (MIC) values of the chloroform extract of endophytic Aspergillus fumigatus ranged from 0.05 to 0.5 mg/ml, and the viable cell count study revealed its bactericidal activity. Mutagenicity and MTT assay disclosed that the crude extract of the said endophyte is neither cytotoxic nor mutagenic showing that it is biosafe [16]. Carbungco et al. [17] isolated a total of 24 endophytic fungi belonging to the genera Fusarium, Xylaria, Pestalotiopsis, Aspergillus, Nigrospora, Stachybotrys, Rhizoctonia, and Macrophomina from M. oleifera leaves. Fungal endophytes of Moringa and their metabolites had not been explored in any locality of Pakistan. The compounds produced by endophytic fungus of M. oleifera could be an alternative source for human welfare. The current study not only aimed at isolating and identifying the endophytic fungi from the leaves of M. oleifera but also searching for the potent yet novel bioactive compounds from this medicinal plant.

Collection of Samples.
Mature trees of M. oleifera (3 to 8 years old) grown in Pakistan Forest Institute, Khyber Pakhtunkhwa, Pakistan were randomly selected for the collection of leaf samples. The collected leaf samples were transferred to the zipper bags, labelled and transported to the microbiology laboratory at Center of Biotechnology and Microbiology, University of Peshawer for further processing. Physical examination was performed for visible symptoms of disease; healthy samples were processed for fungal isolation within few hours after collection.

Isolation of Pure Strains of Endophytic
Fungi. The endophytes were isolated using a modified method described by Arnold [18]. Briefly, leaf samples were carefully washed with sterile water and then surface sterilized with 75% ethanol for 30 seconds, followed by soaking in 2% of sodium hypochlorite (NaOCl) solution for 2 minutes. The leaves were finally rinsed thoroughly with sterile distilled water to remove any traces of the ethanol and NaOCl.
Air dried samples were cut in 1 cm pieces aseptically with the help of sterile scalpel, which were then carefully placed on Sabouraud Dextrose agar (SDA) plates, and incubated at 30°C for 7 days. Chloramphenicol was added in the media before pouring in plates to avoid bacterial growth. The plates were thoroughly checked on a daily basis to monitor the growth of endophytic fungi. After the appearance of the fungal hyphae, they were carefully picked and inoculated on fresh labelled plates and slants of Potato Dextrose Agar. Repeated culturing and subculturing resulted in pure cultures. Fungal cultures were then stored at 4°C till further processing.

Cultivation of Endophytic Fungi and Metabolite
Extraction. The fungus was cultured by placing agar blocks of actively growing pure culture (3 mm in diameter) in a 500 ml Erlenmeyer flask containing 100 g of rice medium. Incubation was done at room temperature for 3 weeks. Afterward, the fermentation was brought to a halt by the introduction of 500 ml of ethyl acetate into the flask. The Whatman No. 1 filter paper was used to separate the fermented mixture. The metabolite from the fungus was extracted by using ethyl acetate as an organic solvent. Ethyl acetate was then evaporated with the help of a rotary evaporator at 50°C, and the resultant compounds were dried in CaCl 2 desiccators to yield the crude metabolites. The crude extract was then kept in an Eppendorf tube at 25°C for bioassays.  [19] was adopted for the morphological identification of various fungal strains isolated from the leaves of M. oleifera. Mycological identification keys and taxonomic description were used to identify the isolated fungi at the genus level [20].

Microscopic
Identification. The morphology of spores as well as mycelia of fungal isolates were studied, recognized, and identified by using the lactophenol blue staining and light microscopy [21]. By using the well diffusion method, the crude ethyl acetate extracts of endophytic fungi suspended in Dimethyl Sulfoxide (DMSO) were screened against bacterial pathogens, and the zones of inhibition were measured [22,23]. Amoxicillin was used as positive control, while dimethyl sulfoxide (DMSO) was used as negative control. Percent inhibition was calculated using following formula: The antifungal activity was performed as per reported procedure of Ahmad et al. [24]. Exactly, 24 mg/ml stock solution of test sample was prepared by using DMSO. Slants preparation was done by using about 5 ml of SDA medium. Then, from stock solution, 66.6 μl of the sample was transferred to test tubes containing 7 days old fungal culture, and the tubes were incubated for 7 days at 25 ± 1°C . After seven days of growth, results were compared with controls, and percent inhibition was calculated. For positive controls, we used Amphotericin-B for C. albicans and Miconazole for other pathogenic fungi [25]. DMSO was used as a negative control.

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BioMed Research International 2.6. Phytotoxic Assay. Phytotoxic properties of the isolated endophytes against Lemna minor were assessed by adopting the established protocol of Rashid et al. [26]. Briefly, Emedium was prepared and autoclaved at 121°C for 15 minutes. Dimethyl sulfoxide (DMSO) was used to make 20 mg/ml of crude extracts as stock solutions. To each sterilized flask, 20 ml of E-medium was added, followed by addition of eighteen L. minor leaves. Paraquat was used as positive control and E-medium without extracts as negative control. The flasks were plugged with sterile cotton and then placed in a plant growth chamber for seven days at 30°C, 9000 Lux and 60% moisture. The fronds were visually examined after seven days of incubation. The percent growth inhibition was calculated as 2.7. Cytotoxic Assay (Brine Shrimp Lethality Assay). The crude extract of fungal metabolites was used to measure the cell lethality against the eggs of brine shrimps (Artemia salina). Marine water was prepared by dissolving 38 g of sea salt in 1000 ml of purified H 2 O. The mixture was filtered, and the pH was set to 7.4 in order to facilitate hatching of the shrimp eggs [25]. In a small plastic hatching chamber, the seawater was kept, having dark and light partitioning. Eggs (1 mg) were placed at the dark/shaded side of the chamber, while the hatched shrimp were attracted by the light and moved to the other side. Larvae per ten shrimps were collected and shifted to flasks containing 5 ml of sea water. In 1% DMSO, the crude extracts were made at 20 mg/mL concentration. Plain marine water was used as a negative control, while Etoposide, a cytotoxic drug, was used as a positive control. Magnifying glass was utilized to count the dead shrimps after 24 hours of incubation period [27], and the cytotoxicity was calculated using the following formula: 2.8. Insecticidal Assay. The direct contact method, also known as the impregnated filter paper method, was used to determine the insecticidal activity of the fungal crude extracts against the Callosobruchus analis, Tribolium Castaneum, and Rhyzopertha dominica. The insect species were obtained from the National Institute of Food and Agriculture, NIFA, Peshawar. Briefly, crude endophytic fungal extracts in methanol were added to the filter paper in petri plates with the help of sterile micropipette. After overnight evaporation, the insects of the same size and age were transferred to the plates. The plates containing insects were incubated overnight in a growth chamber at 27°C; the humidity was maintained by placing a beaker full of water in the growth chamber [28]. Permethrin was used as a positive control, while methanol was used as a negative control.
The percent mortality was calculated as follows:

Results and Discussion
3.1. Isolation of Endophytic Fungi. Incubation of sterile cut leaves of M. oleifera on PDA plates resulted in different colored and textured colonies. Pure isolates were obtained by repeated culturing and subculturing of endophytic fungi based on difference in colonies' margins, pigments, growth rate, and morphology. A total of 22 endophytic fungal strains was isolated from the leaves of M. oleifera.

Characterization of the Isolated Fungal
Endophytes. Isolated endophytic fungi from M. oleifera leaves were identified via morphological (microscopic and macroscopic) characteristics (Table S1 and S2 and Figure S1).  [31]. In fact, most of the endophytes that belong to the above-mentioned genera are already known for the production of bioactive compounds. For example, Penicillium roqueforti harbors andrastins A-D [32], Fusarium oxysporum produces volatile organic compounds [33], and Aspergillus carneus releases depsipeptides, marcfortine A, and aspergillicins A-E [34].
3.4. Antifungal Activity. MOL3, MOL11, MOL17, and MOL20 demonstrated antifungal activity against all the tested fungal strains (Figure 2). MOL1 showed moderate antifungal activity against Candida albicans (46%), Saccharomyces cerevisiae (30%), and Pneumocystic pneumonia (21%), whereas no activity against Verticillium chlamydosporium. The antimicrobial activity of MOL3 against pathogenic C. albicans was 33%, S. cerevisiae was 40%, P. pneumonia was 25%, and V. chlamydosporium was 10%. MOL7 showed higher antifungal activity against C. albicans (56%), low activity against S. cerevisiae (27%) and V. chlamydosporium (23%), but no activity against Pneumocystic pneumonia. MOL11 showed strong antifungal activity against V. chlamydosporium, while moderate to low activity against C. albicans, S. cerevisiae, and P. pneumonia. The effect of MOL17 extracts on percent inhibition against C. albicans was 14%, S. cerevisiae was 13%, P. pneumonia was 36%, and V. chlamydosporium was 30%. Antifungal activity of MOL20 against all 4 tested were noticed, but the activity of crude extract was weak ( Figure 2). Moreover, the extract of the all other isolated endophytes from M. oleifera leaves was inactive against the tested pathogenic strains of fungi (Results not shown). As it is known that endophytes release a broad range of potent compounds, more and more researchers from various fields including health are engaged to evaluate the antimicrobial activity of these fungi [10]. In this perspective, Yu et al. [37] observed antifungal activity of endophytic fungi ty-64 (Oidium sp.) isolated from the Camellia oleifera against the pathogenic C. oleifera anthracnose. Aspergillus terreus is one of the well-studied endophytes that releases hundreds of compounds to support host plant under stress [15]. Quite recently, it was observed that the compounds in the A. terreus exudates can inhibit the growth of mucormycosis causing Syncephalastrum racemosum, Mucor racemosus, and Rhizopus oryzae [38]. The potent antifungal activity of endophytic fungi is due to the presence of cytochalasin alkaloids, terpenoids, polyketides, propanoic acid, piliformic acid, cordycepsidone A, ferulic acid, etc. in their exudates [39].
3.5. Phytotoxic Activity. Focus on endophytic fungal secondary metabolites is growing day by day to discover novel phytotoxic compounds in order to develop bioherbicides. The use of organic weedicides is one of the best substitutes to monitor a vast array of weeds in crops and forests [40][41][42]. In fact, agricultural productivity in terms of crop yield got reduced around the globe, especially in Pakistan because of weeds. Losses in crops' growth and development caused by weeds have ignored over the years; however, weeds harm the agricultural crops more as compared to the insects and pests [43]. In this study, some of the isolated fungi from leaves of M. oleifera exhibited good phytotoxic activity against Lemna minor ( Figure 3). As expected, the metabolites of the potent strains showed high activity at 100 μg/ml concentration as compared to the 10 μg/ml against L. minor. The exudates of isolate MOL3 showed good percent inhibition, i.e., 40% at 10 μg/ml and 50% at 100 μg/ml. MOL7, MOL9, MOL15, MOL17, and MOL19 were among the best strains which inhibited higher than 50% of L. minor growth at 100 μg/ml. MOL1 showed 40% inhibition at 100 μg/ml and 20% at 10 μg/ml. The crude extract of endophytic fungal MOL2 and MOL5 isolates demonstrated very weak phytotoxic activity (Figure 3). The exudates of remaining isolates were found inactive against L. minor (Results not shown). A large number of endophytes have been reported to release nonvolatile as well as volatile compounds that have proven phytotoxic activity against various weeds [44]. In 2016,   3.6. Brine Shrimp Biolethality Assay. Brine shrimp biolethality (BSL) assay was carried out by using crude extracts of endophytic fungi isolated from the leaves of M. oleifera (Figure 4). Similar to phytotoxic assay, the BSL assay demonstrated high activity of potent endophytes at high concentration (10 μg/ml) as compared to the low concentration (5 μg/ ml). Besides this, great diversity (ranged from 10% to 60%) was observed in the extracts of endophytes that showed activity against the shrimps. The fungal extracts, MOL2, MOL6, MOL8, and MOL16, exhibited high toxicity at 10 μg/ml, while at 5 μg/ml same samples showed mild activity. The extract of fungal endophytes MOL3, MOL20, and MOL21 displayed low percent mortality at 10 μg/ml concentration. Quite interestingly, the sample of MOL13 displayed same activity (25%) at 5 and 10 μg/ml (Figure 4). The extract of remaining fungal endophytes isolated from M. oleifera leaves showed no activity in the BSL assay (Results not shown). BSL assay is indeed a basic assay to notice the cytotoxic potential of natural or synthetic drugs; however, for the confirmation of cytotoxicity rigorous experiments, using cell lines is needed. It has been known for years that the synthetic drugs have the capability to fight the cancerous cells, but they possess some unavoiding side effects. To keep health deterring side effects of the synthetic drugs away, an alternative natural remedy with no side effects is required. Endophytes can serve the said purpose very well as it can release Taxol, podophyllotoxin, camptothecin, vinca alkaloids, phomoxanthone a, brefeldin a, altersolanol, macrosporin, etc. [46]. Our results are in close agreement with those of Majoumouo et al. [47], who observed cytotoxic activity in the extracts of Fusarium oxyporum. 3.7. Insecticidal Activity. Extracts from endophytic fungi contain numerous compounds, which play a key role in defending host plant species against insects and pests. In this study, the insecticidal activities of fungal extracts isolated from M. oleifera leaves were examined against Tribolium castaneum, Callosbruches analis, and Rhyzopertha dominica ( Figure 5). High mortality of T. Castaneum was recorded for the extracts of endophytic isolates MOL6 (40%) and MOL19 (40%), whereas MOL12 (30%) showed moderate activity. The extracts of isolates MOL1 (20%), MOL2 (20%), MOL17 (20%), and MOL20 (20%) demonstrated low, and MOL5 (10%), MOL9 (10%), and MOL16 (10%) exhibited very low activity against T. Castaneum. In case of C. analis, 50% of the mortality was caused by the extract of MOL20, followed by MOL2 (30%), MOL17 (30%), and MOL19 (30%). The extracts of MOL3 (20%), MOL5 (20%), and MOL6 (20%) showed weak, and MOL1 (10%), MOL9 (10%), MOL12 (10%), and MOL16 (10%) exhibited very weak anti-C. analis activity. Strong mortality was observed in R. dominica, when they were given the crude extracts of MOL2 (40%), MOL17 (40%), MOL1 (30%), and MOL5 (30%). On the other hand, MOL19 (20%) presented low anti-R. dominica activity, whereas the lowest activity, i.e., 10% mortality was observed in R. dominica fed with MOL3, MOL6, MOL9, MOL12, and MOL20 ( Figure 5). The exudates of remaining isolates were found inactive against the tested insects (Results not shown).
T. Castaneum is a global pest found in the stockpiled goods, mainly food grains [48], while C. analis beetles specifically grow in the legumes, for example, beans [49], and R. dominica is a grain borer of wheat and rice [50]. In general, these pests can severely affect the crop yield and development, so to control such pests, potent pesticides with low effect on the environment are demanded. Shi et al. [51] isolated potent insecticidal compounds from the endophytic fungi Claviceps purpurea. Likewise, Sarocladium strictum was observed to produce sebacic acid, Penta methoxy flavone, Cis-13-octadecenoic acid, and n-hexadecanoic acid that killed the 2nd larval instar of S. littoralis [52].
3.8. Larvicidal Activity. Mosquitoes serve as a vector of many human pathogenic viruses that cause lots of deaths every year around the world [53]. Usage of synthetic insecticides for killing or halting the growth of vector mosquitoes induced physiological resistance [54]. Ae. Aegypti is a

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BioMed Research International primary vector of yellow fever and dengue hemorrhagic fever [55]. In the present study, 200 ppm of MOL3 crude extract has killed 40% of Ae. Aegypti larvae, while MOL2 extract reduced 10% of the larval population at the same concentration ( Figure 6). The extracts of the rest of the isolated endophytes from M. oleifera leaves have no impact on the Ae. Aegypti larvae (Results not shown). The purified compounds in the extract of MOL3 might play a vital role in controlling yellow fever and dengue in the developed and nondeveloped areas of the world. In recent times, pentane, 1,1,1,5-tetrachloro-preg-4-en-3-one, 17. αhydroxy-17. β-cyano-, pentane, 1,1,1,5-tetrachloro-, and trans-3-undecene-1,5-diyne were found in the extract of Aspergillus tamarii that exhibited vigorous activity against Ae. aegypti and Culex quinquefasciatus [56].
3.9. Antileishmanial Activity. Three fungal extracts were subjected for leishmanial activity against Leishmania major (promastigotes). The results in Table 1  isolated from the leaves of exhibited antileishmanial activity. Further investigation of the crude extract showed the presence of cochlioquinone A, isocochlioquinone A, and anhydrocochlioquinone A [57]. In Panama, antileishmanial activity was found in the extract of fungus Edenia sp., which was attributed to the palmarumycin CP17 and palmarumycin CP18 [58].

Conclusion
The above study suggested that M. oleifera plant is a source of novel yet potent endophytic fungi. The crude extracts of   7 BioMed Research International some of the isolated fungal strain from the leaves of M. oleifera showed antimicrobial, antifungal, termicidal, phytotoxic, insecticidal, cytotoxic, and anti-inflammatory activities. The compounds in crude extracts of isolates MOL3 and MOL7 can serve as best antibiotics against E. coli, while that of MOL6 against S. aureus. Similarly, the strains MOL1 and MOL7 can serve as the antifungal agent against C. albicans and MOL11 against V. chlamydosporium. The isolates, MOL3, MOL7, MOL9, MOL15, MOL17, MOL18, and MOL19, can be used as a source of potent herbicide. MOL2 can be used as a source of cytotoxic, MOL2, MOL6, MOL17, MOL19, and MOL20 as insecticidal, and MOL3 as larvicidal and antileishmanial agents. Moreover, further studies are required on the purified compounds present in the crude exudates of the above mentioned potent strains.

Data Availability
All the data are included in the manuscript and supplementary files.

Conflicts of Interest
The authors declare that there is no competing interest of any nature related to this manuscript.